Proposed signal transduction role for conserved CheY residue Thr87, a member of the response regulator active-site quintet

Abstract

CheY serves as a structural prototype for the response regulator proteins of two-component regulatory systems. Functional roles have previously been defined for four of the five highly conserved residues that form the response regulator active site, the exception being the hydroxy amino acid which corresponds to Thr87 in CheY. To investigate the contribution of Thr87 to signaling, we characterized, genetically and biochemically, several cheY mutants with amino acid substitutions at this position. The hydroxyl group appears to be necessary for effective chemotaxis, as a Thr-->Ser substitution was the only one of six tested which retained a Che+ swarm phenotype. Although nonchemotactic, cheY mutants with amino acid substitutions T87A and T87C could generate clockwise flagellar rotation either in the absence of CheZ, a protein that stimulates dephosphorylation of CheY, or when paired with a second site-activating mutation, Asp13-->Lys, demonstrating that a hydroxy amino acid at position 87 is not essential for activation of the flagellar switch. All purified mutant proteins examined phosphorylated efficiently from the CheA kinase in vitro but were impaired in autodephosphorylation. Thus, the mutant CheY proteins are phosphorylated to a greater degree than wild-type CheY yet support less clockwise flagellar rotation. The data imply that Thr87 is important for generating and/or stabilizing the phosphorylation-induced conformational change in CheY. Furthermore, the various position 87 substitutions differentially affected several properties of the mutant proteins. The chemotaxis and autodephosphorylation defects were tightly linked, suggesting common structural elements, whereas the effects on self-catalyzed and CheZ-mediated dephosphorylation of CheY were uncorrelated, suggesting different structural requirements for the two dephosphorylation reactions.

FIG. 1

Ability of CheY Thr87 mutants to display chemotaxis through soft agar. Semisolid agar was stabbed with E. coli KO641 recA containing no plasmid (−) or plasmid pRBB40 encoding wild-type cheY (WT) or cheY mutants with any of the indicated substitutions at position 87. The plate was incubated at 30°C for 12 h.

FIG. 2

Effect of substitution at Thr87 on rotational behavior. E. coli strains harboring plasmids that encode wild-type (WT) or mutant cheY were grown in tryptone broth at 30°C and tethered to glass coverslips with antiflagellar antibodies (5). For each sample, 20 cells were examined for 20 s/cell, and rotational behavior was recorded in one of seven categories from exclusively CCW (CCW) to exclusively CW (CW). The results are depicted as histograms. Columns A to C refer to strain and plasmid background as follows: (A) KO641 recA (ΔcheY) carrying pRBB40 encoding CheY with the indicated amino acid at position 87 as well as CheZ; (B) KO641 recA carrying pRBB40 encoding CheY with both an Asp→Lys substitution at position 13 and the indicated amino acid at position 87 as well as CheZ; (C) RP5231 (ΔcheY-cheZ) carrying pRBB38 encoding CheY alone with the indicated amino acid at position 87. ND, not done.

FIG. 3

Steady-state phosphorylation properties of CheY proteins with substitutions at position 87. Purified CheA kinase (14 pmol) and wild-type (WT) or mutant CheY protein (70 pmol), in the absence (−) or presence (+) of CheZ (14 pmol), were incubated in the presence of 0.3 mM [γ-³²P]ATP as described in Materials and Methods. Reaction products were separated by SDS-PAGE. A phosphorimager scan of a dried gel from one such experiment is shown.

FIG. 4

Autodephosphorylation rates of wild-type and mutant CheY proteins. CheY proteins were phosphorylated by incubation with purified radiolabelled CheA-P (see Materials and Methods), and aliquots were removed to 2× SDS sample buffer at various time points. The proteins were separated on 15% polyacrylamide gels, and dried gels were analyzed by phosphorimaging. The results are plotted as percent phosphorylated CheY remaining at indicated times. The zero time point represents labelling of CheY after a 10-s incubation with CheA-P. CheY proteins are indicated as follows: wild-type CheY (•), CheYT87S (▪), CheYT87A (□), CheYT87C (○), CheYT87I (×).

FIG. 5

Comparison of autodephosphorylation rates and degrees of stimulation by CheZ for mutant CheY proteins containing various amino acids at position 87. (Top) Autodephosphorylation rates were derived from the data of Fig. 4 and are plotted in comparison to wild-type CheY. (Bottom) A series of dephosphorylation experiments similar to those displayed in Fig. 4 were done in the presence of different concentrations of CheZ. The magnitudes of rate enhancement achieved per amount of CheZ added are plotted in comparison to wild-type CheY. WT, wild type.